PROJECT SUMMARY DNA replication stress is induced by the slowing of replication fork progression and can be caused by a variety of exogenous and endogenous factors. These include exogenous factors that damage the genome, such as UV light, alkylating agents and aldehydes, as well as endogenous sources of DNA damage. DNA secondary structures that act as barriers to DNA replication, such as R-loops, G-quadruplexes and other palindromic structures also stall the replication fork, and at least some of these can form as a result of genotoxic stress. Importantly, prolonged fork stalling can lead to double-strand break formation and genome instability, which is associated with cancer and numerous other disorders. A critical aspect of the response to DNA damage is thought to involve the remodeling of replication forks into another secondary structure, the reversed fork. This structure has been speculated to protect the stalled fork and promote repair or bypass of lesions that stall the fork. There is much interest in understanding how often reversed forks and other DNA secondary structures such as R-loops are formed, and how they impact DNA replication. However, methods for the visualization of these intermediates are limited and difficult to access, and in many cases they are non-quantitative or prone to problems based on the population-based nature of the measurements made. In this application, a single- molecule based approach to address this problem and to detect DNA secondary structures in the context of replication will be explored. The approach to be implemented is modular in nature and thus could be applied to different DNA secondary structures. In the first aim, the approach will be developed to detect reversed replication forks, and in the second aim, the approach will be developed to detect R-loops. If successful, this single-molecule approach would allow investigators to quantify R-loops and reversed forks in different cell types and study the impact of these structures on DNA replication fork dynamics. It would also provide a foundation for the development of tools to analyze other DNA secondary structures as well. !